The purpose of my proposed research project is to investigate the mechanisms and regulation of inner stripe of outer medullary collecting tubule (OMiCT) acidification. Specifically, I will investigate 1) the apical and basolateral membrane transport mechanisms for H+/HCO-3, 2) their ionic regulation, and 3) their regulation by hormones, cell calcium activity, and extracellular tonicity. All studies will be performed in the in vitro microperfused OMiCT. In the first part of my project, I plan to examine the transport mechanisms for H+/HCO-3 which are present on the apical and basolateral membranes of the different cell types in the OMiCT, using the measurement of cell pH by epifluorescence. This will allow us to develop assays by which we can examine the regulation of these transporter. In the second part of my project, I will examine the ionic regulatory factors which modulate acidification in the OMiCT. Specifically, I will look at the effects of luminal and peritubular concentration of Na+, K+, C12, and HCO3- on the rate of net H+ transport with flux studies and examine the specific transport mechanisms affected using the epifluorescent measurement of cell pH. Finally, I will examine how H+ transport is regulated by hormones and extracellular tonicity. In this part of my project, I will follow-up on previous studies we have performed demonstrating cyclic AMP stimulation and prostaglandin inhibition of net H+ transport in this segment. Specifically, I will examine the effects of cyclic AMP, prostaglandin E2, aldosterone, protein kinase C activation, intracellular calcium activity, and extracellular tonicity on net H+ transport and examine the specific transport mechanisms involved by using epifluorescent measurement of cell pH. I will also examine how these determinants interact with each other.